Myelofibrosis (MF) is the deadliest of the various myeloproliferative neoplasms (MPN) that affect approximately 150,000 people in the United States. MPN are characterized by abnormal proliferation of one or more of the blood lineages, bone marrow fibrosis, splenomegaly, and progression to acute myeloid leukemia. The identification of recurrent mutations in these patients, the most common being the JAK2V617F mutation found in 95% of polycythemia vera (PV) and 65% of primary MF (PMF) patients, suggests a common molecular pathogenesis centered on aberrant JAK/STAT signaling; however, JAK inhibitors alone offer only modest clinical benefit, without cure. Combinations of JAK inhibitors with other inhibitors of oncogenic pathways such as AKT, MEK/ERK, mTOR, or Hedgehog (Hh), show greater efficacy then JAK inhibitors alone, suggesting that a complex signaling network drives MPN. During my K08 award period, we have demonstrated that: 1) JAK2V617F transgenic mice show increased sonicHh (Shh) ligand expression and activation of Smoothened (Smo)/Hh signaling 2) Treatment with the Hh/Smo inhibitor, PF-04449913 (PF-913, glasdegib), reduces the splenomegaly, cytokine production, marrow fibrosis and JAK2V617F allele burden 3) JAK2V617F transgenic tissues show increased MAPK and NFKB signaling as well as increased TGF-B levels, which decrease with Smo inhibition 4) JAK2V617F mutant cells cause Hh ligand dependent activation of Hh target genes and TGF-B/SMAD2 signaling in stromal cells. Therefore, we hypothesize that Hedgehog signaling is essential for JAK2V617F driven diseases and represents and important therapeutic target. The aims of this study are to determine how JAK2V617F leads to Hh pathway activation and delineate the autocrine vs paracrine effects of abnormal Hh signaling in the bone marrow microenvironment. We will focus on understanding how TGF-B mediates fibrosis and alters the host immune response to MPN. This study will make use of a breakthrough imaging technique known as Imaging Mass Cytometry, to characterize with 40+ parameters the paracrine signaling events and immune response that drives the fibrosis reaction. Understanding the interaction between mutant JAK signaling and hedgehog signaling has implications beyond MPN, as abnormal JAK/STAT and Hedgehog signaling have been implicated in numerous cancers as well as in non-cancerous conditions such as autoimmune diseases, graft verses host disease, inflammation and liver cirrhosis.